RNA interference (RNAi) offers the exciting opportunity to treat disease by knocking down disease-causing genes. Recent early phase clinical trials have shown promising and sustained gene knockdown and/or clinical benefit in a handful of diseases caused by aberrant gene expression in the liver. The major obstacle to harnessing RNAi for cancer treatment is delivery of small RNAs to disseminated cancer cells. Most epithelial cancer cells and the tumor-initiating cells (T-IC) within them highly express EpCAM, the first described tumor antigen. All epithelial breast cancer cell lines we tested stain brightly for EpCAM, while immortalized normal breast epithelial cells and fibroblasts do not. Targeted gene knockdown in epithelial cancer cells in vitro can be achieved using chimeric RNAs composed of a structured RNA, called an aptamer, selected for high affinity binding to EpCAM, that is covalently linked to an siRNA. These EpCAM aptamer-siRNA chimeras (AsiC) are taken up by EpCAM+ cells and selectively cause gene knockdown in epithelial breast cancer cells, but not normal epithelial cells. Moreover knockdown of PLK1 with EpCAM-AsiCs suppresses colony and mammosphere formation of epithelial breast cancer lines, in vitro assays of tumor-initiating potential, and tumor initiation. Subcutaneously injected PLK1 EpCAM-AsiCs are taken up specifically by EpCAM+ basal-A triple negative breast cancer (TNBC) orthotopic xenografts and cause rapid tumor regression. TNBC has the worst prognosis of any breast cancer and there is no targeted therapy for it. The goal of this proposal is to evaluate whether EpCAM-AsiCs can be used for targeted gene knockdown to treat epithelial (basal-like) TNBC cancers, sparing normal cells, and eliminate the T-ICs within them. One goal is to define which breast cancer subtypes can be targeted by EpCAM-AsiCs and determine how EpCAM level affects uptake and gene silencing. Relative uptake/knockdown in cancer cells expressing EpCAM and normal epithelium will be evaluated in human breast cancer tissue explants. Another goal is to determine whether EpCAM-AsiCs can target breast T-ICs to disrupt tumor initiation. An important goal is to optimize the drug-like features of EpCAM-AsiCs to produce a drug candidate suitable for clinical evaluation. To achieve this aim, we will optimize EpCAM-AsiCs for cell uptake, endosomal release, systemic delivery and in vivo gene knockdown. Pharmacokinetics (PK) and pharmacodynamics (PD) of EpCAM-AsiC uptake and gene silencing and tumor suppression will be evaluated using live animal imaging in TNBC cell line xenograft models. As proof of principle, we will evaluate the antitumor effect of knockdown of PLK1, which is needed for cell proliferation. In addition knockdown of novel gene targets identified in a genome-wide siRNA screen for TNBC genetic dependencies will be evaluated in mouse xenograft models. At the end of this program, we will have an optimized EpCAM-AsiC and knowledge of its PK, PD and possible toxicity, to lay the groundwork for formal toxicity and other preclinical studies needed to initiate clinical proof of concept studies.